7.5% western blot gels were loaded with 70ug of both IMR-90 and HCA2 whole cell extracts. The samples had been prepared in Laemmlie buffer and a protease inhibitor cocktail. The antibody was then used in a 1/1000 dilution with a monoclonal goat anti-mouse secondary antibody in a 1/5000 dilution. The antibody worked very well for the HCA2 cells. With a 2 hour incubation in the primary antibody, the resulting picture was a nice strong band at 150 kDa (30 sec exposure). The band is a bit smeared underneath, but this does not get in the way of observing the band. There were not any non-specific bands. The band that appeared in the IMR-90 cells was much weaker, and may be impoved by loading more protein on the gel. Overall I would use this antibody again to study Rad50.
Abcam user community
Submitted Mar 02 2006
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